Over the course of recent years, stain-free histopathology of fresh tissue within minutes has been established by diverse nonlinear optical processes to improve upon conventional histotechnology based on single-photon microscopy. The stain-free non-linear imaging techniques obviate time-consuming paradigmatic elements of standard histology procedures. A summary of various of the nonlinear optical modalities employed is provided by Pavillon et al., “Multimodal label-free microscopy,” J. Innovative Optical Health Sciences, vol. 7, 1330009 (2014), incorporated herein by reference.
Phospholipid-enclosed extracellular vesicles have been the subject of study since the 1980s, however the in vivo role of extracellular vesicles has been elusive because, prior to the present invention, there has been no method for observing them label-free in live tissue.
Techniques are known in the art for imaging media, including biological media, using each of the constituent nonlinear modalities of the current invention, namely second harmonic generation (SHG), third harmonic generation (THG), two-photon auto-fluorescence (2PAF), three-photon auto-fluorescence (3PAF), and coherent anti-Stokes Raman scattering (CARS). Auto-fluorescence arises due to photoexcitation of various molecules naturally occurring in biological matter, with flavins (e.g., flavin adenine dinucleotide (FAD)) and nicotinamide adenine dinucleotide (NADH) notably used as biomarkers in live cells. Integration of coherent anti-Stokes Raman imaging with multiphoton imaging has also been discussed, as by Lu et al., “Integrated Coherent Anti-Stokes Raman Scattering and Multiphoton Microscopy for Biological Imaging using Spectral Filtering of a Femtosecond Laser,” Appl. Phys. Lett., vol. 96, 133701 (2010), which is incorporated herein by reference.
The use of multimodal imaging and CARS in an in-vivo and simultaneous context has been shown by Li et al., “In vivo and simultaneous multimodal imaging: Integrated multiplex coherent anti-Stokes Raman scattering and two photon microscopy,” Appl. Phys. Lett, vol. 97, 223702 (2010), which is incorporated by reference. Another multimodal platform is described by Chen et al., “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Expr., vol. 17, pp. 1282-90 (2009), incorporated herein by reference.
Prior to the present invention, described in detail below, it was believed in the art that imaging in the modalities of THG and autofluorescence were mutually incompatible for simultaneous imaging of biological materials in situ. That is because UV absorption of the third harmonic placed a lower bound on the THG excitation wavelength, while, at an infrared wavelength compatible with THG, the autofluorescence signal for either 2PAF or 3PAF was too weak to achieve a meaningful signal to noise, unless excitation intensities were increased to the point where average incident power leads to biological photodamage. For the foregoing reasons, to the best of the knowledge of the inventors, in-situ imaging of extracellular vesicles has eluded all known techniques, and, in order to unlock the potential of four- or five-mode imaging of the sort taught herein, an invention was required.
One dimension of some embodiments the invention described below concerns a supercontinuum source. Several integrated fiber sources, such as a supercontinuum source using photonic crystal fiber (PCF) pumped with 100-200 fs pulses from Yb3+ fiber lasers, are discussed by Genty et al., “Fiber supercontinuum sources,” J. Opt. Soc. Am. B, vol. 24, pp. 1771-85 (2007), incorporated herein by reference. The use of supercontinuum sources in a context of biophotonics is surveyed by Tu et al., “Coherent Fibers Supercontinuum for Biophotonics,” Laser Photon Rev., vol. 7 (2013), which is incorporated herein by reference.
Over the last decade, two platforms of label-free epi-detected imaging have stood out as viable clinical platforms for multiphoton microscopy: One, demonstrated by Zipfel et al., “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA, vol. 100, pp. 7075-80 (2003), which is incorporated here by reference, simultaneously collects the structural information (noncentrosymmetry) of second-harmonic generation (SHG) and the functional information of two-photon auto-fluorescence (2PAF) excited at a short-wavelength (SW) band of ≤950 nm, and is thus termed “SW-SHG&2PAF imaging.” The other, described by Chu et al., “Multimodal nonlinear spectral microscopy based on a femtosecond Cr: forsterite laser,” Opt. Lett., vol. 26, pp. 1909-11 (2001) (hereinafter, Chu, 2001), incorporated herein by reference, simultaneously collects the structural information of SHG and other structural information (optical heterogeneity) of third-harmonic generation (THG) excited at a long-wavelength (LW) band of 1000 nm, and is thus termed “LW-SHG&THG imaging.”
The former platform has been critically limited by highly nonlinear photodamage, and, to a lesser degree, insufficient structural information when tissue lacks noncentrosymmetry (SHG contrast), while the later platform has been critically limited by the absence of functional information including auto-fluorescence intensity and lifetime. The two platforms complement each other, but have long resisted a synergistic integration to simultaneously collect SHG, THG, and auto-fluorescence signals by one single (single-beam fixed-wavelength) excitation. Their simple combination using sequential SW and LW excitations coupled with sequential signal detections on the same field-of-view, as practiced, for example, in Weigelin et al., “Intravital third harmonic generation microscopy of collective melanoma cell invasion: principles of interface guidance and microvesicle dynamics,” Intravital, vol. 1, pp. 32-43 (2012), incorporated herein by reference, not only increases photodamage risk and prevents rigorous spatial co-registration between sequentially detected signals, but also complicates the corresponding fiber-based endoscope or handheld probe afforded by both platforms. Because THG contrast cannot be generated from the SW-SHG&2PAF platform due to UV absorption by tissue and standard optics, there has long been a profound need to introduce functional information to the LW-SHG&THG platform in order to empower multiphoton microscopy as real-time stain-free histology.
Various treatments of the tumor microenvironment in the prior literature have included a non-reductionist view of cancer, interactions at the tumor-host interface, a wound-healing analogue of tumor development, the concept of “seed and soil”, the bipolar effects of stroma in the tumor “organ”, and the Darwinian (environmental) selection of metastatic tumor cells. Signature events occurring in the tumor microenvironment include: (I) recruitment or infiltration of non-native cells such as immune inflammatory cells and bone-marrow-derived cells, and activation or alteration of fibroblasts (or other native cells for promotion of tumor malignancy and protection from immune attack; (II) a mechanically reorganized extracellular matrix, including degraded basement membrane and rearranged, cross-linked, or fibrotic collagen for enhanced local invasion; (III) angiogenesis and lymphangiogenesis for primary tumor growth and subsequent metastasis; (IV) modulation of stroma by small (<1 μm) tumor-associated extracellular vesicles for pre-conditioning proliferation, invasion, and metastasis of tumor cells; and (V) metabolic switch from energy production to biomass production (biosynthesis), i.e., a reinterpreted Warburg effect that enriches amino acids, nucleotides, and fatty acids, discussed by Vander Heiden et al., “Understanding the Warburg effect: the metabolic requirements of cell proliferation,” Science, vol. 324, pp. 1029-33 (2009), which is incorporated herein by reference.
The metabolic switch has long been masked in oxygen- and nutrient-rich tissue cultures, and has therefore been treated as an adaption to the stressful tumor microenvironment. Each of these tumor microenvironment events has been investigated with exogenous molecular labeling probes to identify biomolecules of interest (rare signaling molecules or bulk substances) within biological samples (cell/tissue cultures, xenografts, dissected specimens, transgenic animals, cancer animal models, living subjects, etc.) under in vitro, ex vivo, intravital, or in vivo conditions. However, the interrelation between the events at the macroscopic scale (I-III) and microscopic scale (IV, V) remains elusive due to the lack of an imaging methodology to observe them in concert, in spatially-resolved ways, and without perturbative labels.
Optical imaging can be a promising approach, suggested, among others, by Weissleder et al., “Shedding light onto live molecular targets,” Nat. Med., vol. 9, pp. 123-28 (2003), incorporated herein by reference, to study this interrelation, as long as the molecular labeling agents, including genetic reporters that may affect the living system through genetic manipulation, are avoided to eliminate unexpected perturbations to the tumor microenvironment.
Also, to retain more authentic physiology during carcinogenesis, imaging should be performed in non-xenograft tissues, and using a reflection mode (epi-) geometry so that these events in the future can be potentially monitored in clinical scenarios to evaluate therapeutic strategies. Along this path, linear microscopy techniques using photoacoustics or optical frequency domain imaging, targeting single-photon absorption or scattering contrast, has visualized angiogenesis and lymphangiogenesis. However, the molecular specificity, which is crucial in the era of molecular oncology, has not been satisfactory using the linear microscopy techniques.
Alternatively, nonlinear microscopy targeting two-photon optical noncentrosymmetry contrast χ(2)SHG and three-photon excited auto-fluorescence contrast AF(3) has imaged reorganized collagen and intrinsic fluorophores, respectively. Similarly, nonlinear microscopy has further visualized tumor cells through two-photon excited auto-fluorescence contrast AF(2) and blood cells/vessels through the molecular vibration contrast of coherent anti-Stokes Raman scattering χ(3)CARS. Thus, previously used cell-tracking fluorescent proteins and injected angiogenesis-revealing agents may be avoided. Also, the χ(3)CARS contrast and three-photon optical heterogeneity contrast χ(3)THG have revealed the metabolic alteration to lipid/protein ratio and the release of extracellular vesicles in tissue, respectively. These scattered efforts call for a multicontrast (multimodal) nonlinear imaging platform that, unlike multimodal platforms of the prior art, is capable of collecting spatially co-registered images with different contrasts that include THG and 3PAF of unlabelled biological moieties.